Immunoadsorption

ABSTRACT

Upon administration of rAAV vectors the humoral immune response (neutralizing antibodies) is the first barrier that needs to be overcome. Surprisingly it was found that by using immunoadsorption for depletion of immunoglobulins from the blood (plasma), subjects can be highly efficiently treated with rAAV vectors, i.e. obtain highly efficient transduction after rAAV vector administration, in spite of the presence of high levels of nAb.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present non-provisional U.S. Patent Application claims priority toEuropean Patent Application No. 16204806.0, filed Dec. 16, 2016, theentire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to the field of recombinant adeno-associated viral(rAAV) vector based gene therapy. In particular, to neutralizingantibodies (immunoglobulins) (nAb) against rAAV in subjects and to theuse of immunoadsorption for depleting immunoglobulins from the bloodprior to rAAV administration.

BACKGROUND OF THE INVENTION

Recombinant adeno-associated virus (rAAV) vectors show great promise forgene therapy in a variety of different genetic disorders. AAV vectorshave an excellent safety profile, as demonstrated in non-clinical andclinical studies. Furthermore, AAV vectors were shown to mediate astable therapeutic transgene expression in several non-clinical studies,and more recently in clinical studies. AAV vectors have been successfulin phase I/II studies for haemophilia B, cystic fibrosis, alpha-1anti-trypsin deficiency, Parkinson disease, Duchenne muscular dystrophyand Leber's congenital amaurosis (Selot et al., Current PharmaceuticalBiotechnology, 2013, 14, 1072-1082). Alipogene tiparvovec (Glybera®,uniQure) has been granted marketing authorization in Europe as a genetherapy for the treatment of lipoprotein lipase deficiency (LPLD). HencerAAV vectors are the gene transfer vectors of choice for the delivery ofgenes in humans in vivo.

One major challenge for a successful administration of AAV vector is thepresence of neutralizing antibodies (immunoglobulins) (nAb) that havedeveloped following exposure to wild-type AAV or AAV-based vectors. Inboth cases, the neutralizing serotype-specific antibodies directedtowards the viral capsid proteins reduce the efficiency of gene transferwith AAV of the same serotype.

The current practice in the clinic with regard to pre-existing immunityinvolves the screening of patients for exclusion should patients haveneutralizing antibodies against the AAV capsid (Brimble et al. ExpertOpin Biol Ther 2016, 16(1):79-92 and Boutin et al. Hum Gene Ther 2010,21:704-712). Immunosupressive regimens have been tried in order toreduce the formation of nAb upon first administration to allow for asecond administration (Corti et al., Mol Ther—Meth Clin Dev (2014) 1,14033; Mingozzi et al. Mol Ther vol. 20 no. 7, 1410-1416; McIntosh etal. Gene Ther 2012, 19, 78-85)).

Furthermore, strategies have been suggested to overcome pre-existingantibodies which include plasma exchange and the use ofimmunosuppressive regimens. Plasma exchange strategies involve theremoval of plasma from the blood and exchanging it for plasma notcontaining neutralizing antibodies (e.g. from a donor or the subjectitself) or a defined replacement fluid (Chicoine et al., Mol Ther 2014,vol. 22 no. 2 338-347; Hurlbut et al. Mol Ther 2010, vol. 18 no. 111983-1984). Immunosuppressive regimens aimed at reducing nAb includerituximab combined with Cyclosporin A (Mingozzi et al. Mol Ther vol. 20no. 7, 1410-1416). Such strategies have been tested in animal modelsobtaining limited success, being somewhat effective in subjects havinglow nAb titers. In view of these results it has been suggested tocombine different strategies and/or use a higher rAAV vector dose(tenfold) in order to overcome pre-existing antibodies and achieveeffective transduction of e.g. the liver (Hurlbut et al. Mol Ther 2010,vol. 18 no. 11 1983-1984; Mingozzi et al. Mol Ther vol. 20 no. 7,1410-1416).

Hence, there is a need in the art for strategies that allowadministration of rAAV vectors in subjects that have, or may besuspected to have, neutralizing antibodies.

BRIEF DESCRIPTION OF THE INVENTION

Upon administration of rAAV vectors the humoral immune response, i.e.neutralizing antibodies, is the first barrier that needs to be overcome.The second barrier that needs to be overcome is the cellular response,i.e. once rAAV has been delivered to the target cell transduced cellsmay be eliminated because these present antigens derived from the rAAVcapsid protein and/or the expressed transgene. The second barrier, i.e.the cellular response to AAV capsids, is transient in nature and may becontrolled by monitoring subjects that have underwent rAAV gene therapytreatment and/or by immunosuppressive treatments. As outlined above,strategies that have been employed that are aimed at overcoming thefirst barrier which included the use of plasma exchange and/orpharmaceutical interventions. Strategies employed were cumbersome andhad limited success. The current inventors now provide for a new andimproved means of avoiding this first barrier. Surprisingly it was foundthat by using immunoadsorption for depletion of immunoglobulins from theblood (plasma), subjects can be highly efficiently treated with rAAVvectors, i.e. obtain highly efficient transduction after rAAV vectoradministration, in spite of these subjects having initially high levelsof nAb. By said immunoadsorption, a therapeutic window is provided thattransiently reduces nAb levels long enough to allow rAAV delivery to thetarget cells without severe risks for the subject being treated. This isbecause immunoadsorption is a relatively mild treatment especially whencompared with proposed and tested prior art methods. Also, the cellularresponse can depend on the dose of rAAV being administered. Withoutbeing bound by theory, because the dose of rAAV that needs to beadministered can be lower to achieve efficient levels of transduction bythe invention, not only the first barrier of the humoral response may beovercome, but it may also contribute to reducing or even avoiding thesecond barrier.

Hence, according to the invention, such immunoadsorption methods caneither involve non-specific or specific removal of neutralizing rAAVimmunoglobulins and such immunoadsorption methods are employed outsideof the body, i.e. extracorporeal. Accordingly, in a first embodiment anrAAV vector is provided for use in the treatment of a subject, whereinsaid subject has been subjected to extracorporeal depletion ofimmunoglobulins using immunoadsorption prior to administration of saidrAAV vector.

Also, methods are provided for reducing the anti-rAAV immunoglobulinconcentration in the blood, comprising the steps of:

-   -   a) providing blood;    -   b) providing a device for immunoadsorption;    -   c) separating the blood provided in a) in plasma components and        cellular components;    -   d) subjecting the plasma components obtained in c) to        immunoadsorption by using the device provided in b);    -   e) reconstituting the blood by combining the cellular components        obtained in c) with the plasma components subjected to        immunoadsorption obtained in d).

Preferably, said reconstituted blood is to be administered to a subjectsuch that the blood of the subject has a reduced anti-rAAVimmunoglobulin concentration in the blood. Subsequently, said subjectcan have rAAV administered.

DEFINITIONS

An “rAAV vector” refers to a recombinant adeno-associated virus (AAV)vector which is derived from the wild type AAV by using molecularmethods. An rAAV vector is distinguished from a wild type (wt)AAVvector, since at least a part of the viral genome has been replaced witha transgene, which is a non-native nucleic acid with respect to the AAVnucleic acid sequence as further defined herein. Wild type AAV belongsto the genus Dependovirus, which in turn belongs to the subfamily of theParvovirinae, also referred to as parvoviruses, which are capable ofinfecting vertebrates. Parvovirinae belong to family of small DNA animalviruses, i.e. the Parvoviridae family. As may be deduced from the nameof their genus, members of the Dependovirus are unique in that theyusually require coinfection with a helper virus such as adenovirus orherpes virus for productive infection in cell culture. The genusDependovirus includes AAV, which normally infects humans (e.g.,serotypes 1, 2, 3A, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) or primates(e.g., serotypes 1 and 4), and related viruses that infect otherwarm-blooded animals (e.g., bovine, canine, equine, and ovineadeno-associated viruses). Further information on parvoviruses and othermembers of the Parvoviridae is described in Kenneth I. Berns,“Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FieldsVirology (3d Ed. 1996). For convenience the present invention is furtherexemplified and described herein by reference to AAV. It is howeverunderstood that the invention is not limited to AAV but may equally beapplied to other parvoviruses.

The genomic organization of all known AAV serotypes is very similar. Thegenome of AAV is a linear, single-stranded DNA molecule that is lessthan about 5,000 nucleotides (nt) in length. Inverted terminal repeats(ITRs) flank the unique coding nucleotide sequences for thenon-structural replication (Rep) proteins and the structural (VP)proteins. The VP proteins (VP1, -2 and -3) form the capsid or proteinshell. The terminal 145 nt are self-complementary and are organized sothat an energetically stable intramolecular duplex forming a T-shapedhairpin may be formed. These hairpin structures function as an originfor viral DNA replication, serving as primers for the cellular DNApolymerase complex. Following wtAAV infection in mammalian cells the Repgenes (i.e. Rep78 and Rep52) are expressed from the P5 promoter and theP19 promoter, respectively and both Rep proteins have a function in thereplication of the viral genome. A splicing event in the Rep ORF resultsin the expression of actually four Rep proteins (i.e. Rep78, Rep68,Rep52 and Rep40). However, it has been shown that the unspliced mRNA,encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient forAAV vector production. wtAAV infection in mammalian cells relies for thecapsid proteins production on a combination of alternate usage of twosplice acceptor sites and the suboptimal utilization of an ACGinitiation codon for VP2.

An rAAV vector may have one or preferably all wild type AAV genesdeleted, but may still comprise functional ITR nucleic acid sequences.Preferably, the rAAV vector does not comprise any nucleotide sequencesencoding viral proteins, such as the rep (replication) or cap (capsid)genes of AAV. At least one functional ITR sequence is necessary for thereplication, rescue and packaging of vector genomes into AAV virions.The ITR sequences may be wild type sequences or may have at least 80%,85%, 90%, 95, or 100% sequence identity with wild type sequences or maybe altered by for example in insertion, mutation, deletion orsubstitution of nucleotides, as long as they remain functional. In thiscontext, functionality refers to the ability to direct packaging of thegenome into the capsid shell and then allow for expression in the hostcell to be transduced or target cell. Typically, the inverted terminalrepeats of the wild type AAV genome are retained in the rAAV vector. TheITRs can be cloned from the AAV viral genome or excised from a vectorcomprising the AAV ITRs. The ITR nucleotide sequences can be eitherligated at either end to a transgene as defined herein using standardmolecular biology techniques, or the wild type AAV sequence between theITRs can be replaced with the desired nucleotide sequence. The rAAVvector preferably comprises at least the nucleotide sequences of theinverted terminal repeat regions (ITR) of one of the AAV serotypes, ornucleotide sequences substantially identical thereto, and at least onenucleotide sequence encoding a therapeutic protein (under control of asuitable regulatory element) inserted between the two ITRs. The majorityof currently used rAAV vectors use the ITR sequences from AAV serotype2. Preferred ITR sequences are ITRs from the AAV serotypes 2. A rAAVgenome can comprise single stranded or double stranded(self-complementary) DNA. The single stranded nucleic acid molecule iseither sense or antisense strand, as both polarities are equally capableof gene expression. Single stranded rAAV vectors may utilize thewild-type AAV ITR sequences, such as for example wild-type AAV2 ITRsequences, and double stranded (self-complementary) rAAV vectors mayutilize a modified version of the ITRs, such as for example disclosed inInternational patent application with the publication number WO01/92551. The rAAV vector may further comprise a marker or reportergene, such as a gene for example encoding an antibiotic resistance gene,a fluorescent protein (e.g., gfp) or a gene encoding a chemically,enzymatically or otherwise detectable and/or selectable product (e.g.,lacZ, aph, etc.) known in the art.

The rAAV vector, including all combinations of AAV serotype capsid andAAV genome ITRs, is produced using methods known in the art, asdescribed in Pan et al. (J. of Virology (1999) 73: 3410-3417), Clark etal. (Human Gene Therapy (1999) 10: 1031-1039), Wang et al. (Methods Mol.Biol. (2011) 807: 361-404) and Grimm (Methods (2002) 28(2): 146-157),which are incorporated herein by reference. In short, the methodsgenerally involve (a) the introduction of the rAAV genome construct intoa host cell, (b) the introduction of an AAV helper construct into thehost cell, wherein the helper construct comprises the viral functionsmissing from the wild type rAAV genome and (c) introducing a helpervirus construct into the host cell. All functions for rAAV vectorreplication and packaging need to be present, to achieve replication andpackaging of the rAAV genome into rAAV vectors. The introduction intothe host cell can be carried out using standard molecular biologytechniques and can be simultaneously or sequentially. Finally, the hostcells are cultured to produce rAAV vectors and are purified usingstandard techniques such as CsCl gradients (Xiao et al. 1996, J. Virol.70: 8098-8108). The purified rAAV vector is then ready for use in themethods. High titres of more than 10¹² particles per ml and high purity(free of detectable helper and wild type viruses) can be achieved (Clarket al. supra and Flotte et al. 1995, Gene Ther. 2: 29-37). The totalsize of the transgene inserted into the rAAV vector between the ITRregions is generally smaller than 5 kilobases (kb) in size.

Alternatively, rAAV can be produced in a baculovirus system (BEVS). Theinitial baculovirus system for production of rAAV was described by Urabeet al (Urabe et al. [2002] Human Gene Therapy 13(16):1935-1943) andconsists of three baculoviruses, namely Bac-Rep, Bac-cap and Bac-vec,co-infection of which into insect cells e.g. SF9 resulted in generationof rAAV. The properties of such produced rAAV, i.e. physical andmolecular characteristic including potency, did not differ significantlyfrom the rAAV generated in mammalian cells (Urabe [2002] supra). Inorder to accomplish efficient generation of rAAV vectors in insect cellsthe AAV proteins needed for the process had to be expressed atappropriate levels. This required a number of adaptations of operonsencoding for Rep and Cap proteins. Wild type AAV expresses large Rep78to small Rep52 from two distinct promoters p5 and p19 respectively andsplicing of the two messengers results in generation of Rep68 and Rep52variants. This operon organization results in limited expression ofRep78 and relatively higher expression of Rep52. In order to mimic thelow 78 to 52 ratio Urabe and colleagues constructed a DNA cassette inwhich expression of Rep78 was driven by the partially deleted promoterfor the immediate-early 1 gene (ΔIE-1) whereas Rep52 expression wascontrolled by a strong polyhedrin promoter (polh). The spliced variantsof large and small Reps were not observed in insect cells which likelyrelates to the difference in splicing processes between mammalian andinsect cells. Another technical challenge to be overcome was related tothe expression of the three major viral proteins (VP's). Wild type AAVexpresses VP1, 2 and 3 from p40 promoter. Arising messenger RNA isspliced into two species: one responsible for VP1 expression whereas thesecond expresses both VP2 and VP3 via a “leaky ribosomal scanningmechanism” where the protein is initiated from non-canonical start i.e.ACG, is occasionally missed by the ribosome complex which then proceedsfurther until it finds the canonical start of VP3. Due to thedifferences in splicing machinery between vertebrate and insect cellsthe above described mechanism did not result in generation of propercapsids in insect cells. Urabe et al., decided to introduce amodification of translational start of VP1 which was similar to thesefound in the VP2 in such a way that the translational start of VP1 waschanged to ACG and the initiation context, which consists of 9nucleotides preceeding VP1, was changed to those preceeding VP2. Thesegenetic alterations resulted in expression of the three VPs in thecorrect stoichiometry that could properly assemble into capsids from asingle polycistronic mRNA. The transgene cassette on the other hand wassimilar to what was previously described for mammalian based systems,flanked by ITRs as the only in trans required elements for replicationand packaging. The initial baculovirus system by Urabe (2002, supra) hasbeen further developed see e.g., Kohlbrenner et al. (2005) MolecularTherapy 12 (6):1217-1225; Urabe et al. (2006) Journal of Virology80(4):1874-1885; WO 2007/046703; WO 2007/148971; WO 2009/014445 and WO2009/104964.

The sequence encoding the capsid protein can be a capsid sequence asfound in nature such as for example of AAV2, AAV5 and AAV8.Alternatively, the sequence can be man-made, for example, the sequencemay be a hybrid form or may be codon optimized form, such as for exampleby codon usage of AcmNPv or Spodoptera frugiperda. For example, thecapsid sequence may be composed of the VP2 and VP3 sequences of oneserotype, such as for example AAV1, whereas the remainder of the VP1sequence is of another serotype, such as for example AAV5. The man-madesequence may result of rational design or directed evolutionexperiments. This can include generation of capsid libraries via DNAshuffling, error prone PCR, bioinformatic rational design, sitesaturated mutagenesis. Resulting capsids are based on the existingserotypes but contain various amino acid or nucleotide changes thatimprove the features of such capsids. The resulting capsids can be acombination of various parts of existing serotypes, “shuffled capsids”or contain completely novel changes, i.e. additions, deletions orsubstitutions of one or more amino acids or nucleotides, organized ingroups or spread over the whole length of gene or protein. See forexample Schaffer and Maheshri; Proceedings of the 26th AnnualInternational Conference of the IEEE EMBS San Francisco, Calif., USA to;Sep. 1-5, 2004, pages 3520-3523; Asuri et al. (2012) Molecular Therapy20(2):329-3389; Lisowski et al. (2014) Nature 506(7488):382-386, hereinincorporated by reference.

In the context of the present invention a capsid protein shell may be ofa different serotype than the rAAV vector genome ITR. An rAAV vector ofthe invention may thus be encapsidated by a capsid protein shell, i.e.the icosahedral capsid, which comprises capsid proteins (VP1, VP2,and/or VP3) of one AAV serotype, such as for example AAV serotype 5,whereas the ITRs sequences contained in that rAAV vector may be ofanother serotype, for example any of the rAAV serotypes described above,including a rAAV5 vector. In a preferred embodiment, an rAAV vector isencapsidated by a capsid protein shell of AAV serotype 5 or AAV serotype2 or AAV serotype 8 wherein the rAAV genome or ITRs present in said rAAVvector are derived from AAV serotype 2 or AAV serotype 5 or AAV serotype8. In this embodiment it is preferred that the rAAV vector isencapsidated by a capsid protein shell of the AAV serotype 5 and therAAV genome or ITRs present in said vector are derived from AAV serotype2. In another embodiment, it is preferred that the rAAV vector isencapsidated by a capsid protein shell of the AAV serotype 2 and therAAV genome or ITRs present in said vector is derived from AAV serotype2.

The complete genome of AAV5 and other AAV serotypes has been sequenced(Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, p 1309-1319) andthe nucleotide sequence is available in GenBank (Accession No. AF085716;23 Feb. 2015). The ITR nucleotide sequences of AAV5 are thus readilyavailable to a skilled person. The complete genome of AAV2 is availablein NCBI (NCBI Reference Sequence NC_001401.2; 2 Dec. 2014). They can beeither cloned or made by chemical synthesis as known in the art, usingfor example an oligonucleotide synthesizer as supplied e.g., by AppliedBiosystems Inc. (Fosters, Calif., USA) or by standard molecular biologytechniques.

A “serotype” is traditionally defined on the basis of a lack ofcross-reactivity between antibodies to one virus as compared to anothervirus. Such cross-reactivity differences are usually due to differencesin capsid protein sequences/antigenic determinants (e.g., due to VP1,VP2, and/or VP3 sequence differences of AAV serotypes). Under thetraditional definition, a serotype means that the virus of interest hasbeen tested against serum specific for all existing and characterizedserotypes for neutralizing activity and no antibodies have been foundthat neutralize the virus of interest. As more naturally occurring virusisolates are discovered and capsid mutants generated, there may or maynot be serological differences with any of the currently existingserotypes. Thus, in cases where the new AAV has no serologicaldifference, this new AAV would be a subgroup or variant of thecorresponding serotype. In many cases, serology testing for neutralizingactivity has yet to be performed on mutant viruses with capsid sequencemodifications to determine if they are of another serotype according tothe traditional definition of serotype. Accordingly, for the sake ofconvenience and to avoid repetition, the term “serotype” broadly refersto both serologically distinct viruses (e.g., AAV) as well as viruses(e.g., AAV) that are not serologically distinct that may be within asubgroup or variant of a given serotype. By way of example, rAAV vectorinclude various naturally and non-naturally occurring serotypes. Suchnon-limiting serotypes include AAV-1, -2, -3, -4, -5, -6, -7, -8, -9,-10, -11, -rh74, -rh10, AAV-DJ and AAV-2i8. Again, for the sake ofconvenience, serotypes include AAV with capsid sequence modificationsthat have not been fully characterized as being a distinct serotype, andmay in fact actually constitute a subgroup or variant of a knownserotype.

The term “transgene” is used to refer to a non-native nucleic acid withrespect to the AAV nucleic acid sequence. It is used to refer to apolynucleotide that can be introduced into a cell or organism.Transgenes include any polynucleotide, such as a gene that encodes apolypeptide or protein, a polynucleotide that is transcribed into aninhibitory polynucleotide, or a polynucleotide that is not transcribed(e.g., lacks an expression control element, such as a promoter thatdrives transcription). A transgene of the invention may comprise atleast two nucleotide sequences each being different or encoding fordifferent therapeutic molecules. The at least two different nucleotidesequences may be linked by an IRES (internal ribosome entry sites)element, providing a bicistronic transcript under control of a singlepromoter. Suitable IRES elements are described in e.g., Hsieh et al.(1995, Biochemical Biophys. Res. Commun. 214:910-917). Furthermore, theat least two different nucleotide sequences encoding for different(therapeutic) polypeptides or proteins may be linked by a viral 2Asequence to allow for efficient expression of both transgenes from asingle promoter. Examples of 2A sequences include foot and mouth diseasevirus, equine rhinitis A virus, Thosea asigna virus and porcineteschovirus-1 (Kim et al., PLoS One (2011) 6(4): e18556). A transgene ispreferably inserted within the rAAV genome or between ITR sequences asindicated above. A transgene may also be an expression constructcomprising an expression regulatory element such as a promoter ortranscription regulatory sequence operably linked to a coding sequenceand a 3′ termination sequence.

In a cell having a transgene, the transgene has beenintroduced/transferred/transduced by rAAV “transduction” of the cell. Acell or progeny thereof into which the transgene has been introduced isreferred to as a “transduced” cell. Typically, a transgene is includedin progeny of the transduced cell or becomes a part of the organism thatdevelops from the cell. Accordingly, a “transduced” cell (e.g., in amammal, such as a cell or tissue or organ cell), means a genetic changein a cell following incorporation of an exogenous molecule, for example,a polynucleotide or protein (e.g., a transgene) into the cell. Thus, a“transduced” cell is a cell into which, or a progeny thereof in which anexogenous molecule has been introduced, for example. The cell(s) can bepropagated and the introduced protein expressed, or nucleic acidtranscribed.

“Transduction” refers to the transfer of a transgene into a recipienthost cell by a viral vector. Transduction of a target cell by an rAAVvector of the invention leads to transfer of the transgene contained inthat vector into the transduced cell. “Host cell” or “target cell”refers to the cell into which the DNA delivery takes place, such as thesynoviocytes or synovial cells of an individual. AAV vectors are able totransduce both dividing and non-dividing cells.

“Gene” or “coding sequence” refers to a DNA or RNA region which“encodes” a particular protein. A coding sequence is transcribed (DNA)and translated (RNA) into a polypeptide when placed under the control ofan appropriate regulatory region, such as a promoter. A gene maycomprise several operably linked fragments, such as a promoter, a 5′leader sequence, an intron, a coding sequence and a 3′ nontranslatedsequence, comprising a polyadenylation site or a signal sequence. Achimeric or recombinant gene is a gene not normally found in nature,such as a gene in which for example the promoter is not associated innature with part or all of the transcribed DNA region. “Expression of agene” refers to the process wherein a gene is transcribed into an RNAand/or translated into an active protein.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide (or polypeptide) elements in a functional relationship. Anucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, atranscription regulatory sequence is operably linked to a codingsequence if it affects the transcription of the coding sequence.“Operably linked” means that the DNA sequences being linked aretypically contiguous and, where necessary to join two protein encodingregions, contiguous and in reading frame.

“Expression control sequence” refers to a nucleic acid sequence thatregulates the expression of a nucleotide sequence to which it isoperably linked.

An expression control sequence is “operably linked” to a nucleotidesequence when the expression control sequence controls and regulates thetranscription and/or the translation of the nucleotide sequence. Thus,an expression control sequence can include promoters, enhancers,internal ribosome entry sites (IRES), transcription terminators, a startcodon in front of a protein-encoding gene, splicing signal for introns,and stop codons. The term “expression control sequence” is intended toinclude, at a minimum, a sequence whose presence is designed toinfluence expression, and can also include additional advantageouscomponents. For example, leader sequences and fusion partner sequencesare expression control sequences. The term can also include the designof the nucleic acid sequence such that undesirable, potential initiationcodons in and out of frame, are removed from the sequence. It can alsoinclude the design of the nucleic acid sequence such that undesirablepotential splice sites are removed. It includes sequences orpolyadenylation sequences (pA) which direct the addition of a polyAtail, i.e., a string of adenine residues at the 3′-end of a mRNA,sequences referred to as polyA sequences. It also can be designed toenhance mRNA stability. Expression control sequences which affect thetranscription and translation stability, e.g., promoters, as well assequences which effect the translation, e.g., Kozak sequences, are knownin insect cells. Expression control sequences can be of such nature asto modulate the nucleotide sequence to which it is operably linked suchthat lower expression levels or higher expression levels are achieved.

As used herein, the term “promoter” or “transcription regulatorysequence” refers to a nucleic acid fragment that functions to controlthe transcription of one or more coding sequences, and is locatedupstream with respect to the direction of transcription of thetranscription initiation site of the coding sequence, and isstructurally identified by the presence of a binding site forDNA-dependent RNA polymerase, transcription initiation sites and anyother DNA sequences, including, but not limited to transcription factorbinding sites, repressor and activator protein binding sites, and anyother sequences of nucleotides known to one of skill in the art to actdirectly or indirectly to regulate the amount of transcription from thepromoter. A “constitutive” promoter is a promoter that is active in mosttissues under most physiological and developmental conditions. An“inducible” promoter is a promoter that is physiologically ordevelopmentally regulated, e.g., by the application of a chemicalinducer. A “tissue specific” promoter is preferentially active inspecific types of tissues or cells. The selection of an appropriatepromoter sequence generally depends upon the host cell selected for theexpression of a DNA segment. Alternatively, a transgene is to beoperably linked to a promoter that allows for efficient systemicexpression. Suitable promoter sequences are CMV (cytomegalovirus)promoter, CBA (chicken beta actin), or liver specific promoters such ashuman alpha-1 anti-trypsin (hAAT) or TBG (thyroxine binding globulin).

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms, depending on the length of the twosequences. Sequences of similar lengths are preferably aligned using aglobal alignment algorithm (e.g. Needleman Wunsch) which aligns thesequences optimally over the entire length, while sequences ofsubstantially different lengths are preferably aligned using a localalignment algorithm (e.g. Smith Waterman). Sequences may then bereferred to as “substantially identical” or “essentially similar” whenthey (when optimally aligned by for example the programs GAP or BESTFITusing default parameters) share at least a certain minimal percentage ofsequence identity (as defined below). GAP uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength (full length), maximizing the number of matches and minimizingthe number of gaps. A global alignment is suitably used to determinesequence identity when the two sequences have similar lengths.Generally, the GAP default parameters are used, with a gap creationpenalty=50 (nucleotides)/8 (proteins) and gap extension penalty=3(nucleotides)/2 (proteins). For nucleotides the default scoring matrixused is nwsgapdna and for proteins the default scoring matrix isBlosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequencealignments and scores for percentage sequence identity may be determinedusing computer programs, such as the GCG Wisconsin Package, Version10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego,Calif. 92121-3752 USA, or using open source software, such as theprogram “needle” (using the global Needleman Wunsch algorithm) or“water” (using the local Smith Waterman algorithm) in EmbossWIN version2.10.0, using the same parameters as for GAP above, or using the defaultsettings (both for ‘needle’ and for ‘water’ and both for protein and forDNA alignments, the default Gap opening penalty is 10.0 and the defaultgap extension penalty is 0.5; default scoring matrices are Blossum62 forproteins and DNAFull for DNA). When sequences have substantiallydifferent overall lengths, local alignments, such as those using theSmith Waterman algorithm, are preferred. Alternatively, percentagesimilarity or identity may be determined by searching against publicdatabases, using algorithms such as FASTA, BLAST, etc.

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. Preferredconservative substitutions for each of the naturally occurring aminoacids are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp toGlu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asnor Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu;Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trpto Tyr; Tyr to Trp or Phe; and, Val to Ile or Leu.

As used herein, “gene therapy” is the insertion of nucleic acidsequences (e.g., a transgene as defined herein) into an individual'scells and/or tissues to treat a disease. The transgene can be afunctional mutant allele that replaces or supplements a defective one.Gene therapy also includes insertion of transgene that are inhibitory innature, i.e., that inhibit, decrease or reduce expression, activity orfunction of an endogenous gene or protein, such as an undesirable oraberrant (e.g., pathogenic) gene or protein. Such transgenes may beexogenous. An exogenous molecule or sequence is understood to bemolecule or sequence not normally occurring in the cell, tissue and/orindividual to be treated. Both acquired and congenital diseases areamenable to gene therapy.

A “therapeutic polypeptide” or “therapeutic protein” is to be understoodherein as a polypeptide or protein that can have a beneficial effect onan individual, preferably said individual is a human, more preferablysaid human suffers from a disease. Such therapeutic polypeptide may beselected from, but is not limited to, the group consisting of an enzyme,a cytokine, an antibody, a growth factor, a hormone and ananti-inflammatory protein.

A “therapeutically-effective” amount as used herein is an amount that issufficient to alleviate (e.g., mitigate, decrease, reduce) at least oneof the symptoms associated with a disease state. Alternatively stated, a“therapeutically-effective” amount is an amount that is sufficient toprovide some improvement in the condition of the individual.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded.

In addition, reference to an element by the indefinite article “a” or“an” does not exclude the possibility that more than one of the elementis present, unless the context clearly requires that there be one andonly one of the elements. The indefinite article “a” or “an” thususually means “at least one”.

The word “approximately” or “about” when used in association with anumerical value (approximately 10, about 10) preferably means that thevalue may be the given value of 10 more or less 10% of the value.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant adeno-associated virus (rAAV) vector based gene therapy hasproven to be successful in treating genetic disorders in clinicalsettings. However, upon administration of rAAV vectors, antibodies (alsoreferred to as immunoglobulins) that are capable of neutralizing rAAVvector can have a dramatic impact on transduction. Such antibodies canbe present when a subject selected for gene therapy has been infectedwith wild-type AAV earlier. Therefore, in a clinical setting the currentpractice is to test subjects for the presence of neutralizing antibodiesprior to rAAV vector administration in order to avoid suchneutralization. Hence, the presence of neutralizing antibodies is anexclusion criterion in current gene therapy treatments with AAV. Forexample, when a subject has neutralizing antibodies for AAV8, e.g.because said subject has previously been infected by wildtype AAV8, sucha subject is excluded from a treatment with an rAAV8 vector. It isunderstood that the rAAV vector that is used may not necessarily have awild-type capsid composed of wild-type VP proteins but may also beconstituted from VP proteins that are derived from a wild-type proteinand thus may not have an exact wild-type sequence. Nevertheless, nAb maybe detected against capsids derived from a wild-type capsid. For manyindications it is envisioned that subjects may have to undergo more thanone gene therapy treatment in their life time. Because a firstadministration with an rAAV vector in a naïve subject generally canresult in triggering neutralizing antibodies, a subsequent treatmentwith the same or similar rAAV vector would not be successful due toneutralizing antibodies that were induced in response to the priortreatment with rAAV which neutralizing antibodies may recognize the rAAVvector to be used in the second treatment.

To overcome this problem, it has been suggested to switch capsids, e.g.use a different serotype, for any subsequent treatment to avoidneutralizing antibodies. Avoiding neutralizing antibodies cannot beregarded to be a solution because suitable alternative serotypes are notalways generally available. Such alternative serotypes or capsids arerequired to have the same tropism, and said subjects need to have anaïve immunity for the alternative capsid or serotype. Hence, as said,other strategies in the prior art that have been suggested to overcomepre-existing antibodies include plasma exchange and the use ofimmunosuppressive regimens. Such strategies have been tested in animalmodels with limited success, being somewhat effective in subjects havinglow nAb titers.

Surprisingly, the current inventors have now found that subjects withneutralizing rAAV antibody titers in the blood, which include subjectswith high titer neutralizing antibodies, can have rAAV administeredafter such subjects are treated with an extracorporeal treatment of theblood that depletes immunoglobulins using immunoadsorption. Withoutbeing bound by theory, such depletion of immunoglobulins byimmunoadsorption may also be advantageous in any subject that is to betreated with an rAAV vector, regardless of whether neutralizingantibodies have been detected prior to said immunoadsorption or not.Hence, in one embodiment an rAAV vector is provided for use in thetreatment of a subject, wherein said subject has been subjected to anextracorporeal depletion of immunoglobulins using immunoadsorption priorto administration of said rAAV vector. To phrase differently, inaccordance with the invention an rAAV vector for use in the treatment ofa subject is provided, wherein said subject has been subjected to anextracorporeal immunoadsorption of immunoglobulins prior toadministration of said rAAV vector. It is understood that with regard tosuitable subjects, these comprise preferably mammals, such as a mammalselected from the group consisting of humans, non-human primates (suchas for examples apes, gibbons, gorillas, chimpanzees, orangutans,macaques), domestic animals (such as for example dogs and cats), farmanimals (such as for example poultry such as chickens and ducks, horses,cows, goats, sheep, pigs). More preferably, such a mammal is a primate.In a more preferred embodiment, the subject is a human.

Immunoadsorption in accordance with the invention is defined as aprocedure that removes immunoglobulins from a body fluid such as theblood. Immunoadsorption involves the use of a binding moiety that iscapable of binding immunoglobulins. Said immunoglobulins bound to thebinding moiety allows to separate the immunoglobulins from the blood,preferably from the blood plasma or serum. Immunoadsorption hence canselectively remove immunoglobulins from the blood while retainingsubstantially the same composition thereof.

It is understood that with an extracorporeal depletion ofimmunoglobulins is meant a treatment of a body fluid that is outside ofthe subject's body. Such an extracorporeal depletion of immunoglobulinscan involve a depletion of immunoglobulins from the blood. Such anextracorporeal depletion of immunoglobulins can also involve a depletionof immunoglobulins from cerebrospinal fluid (CSF). For example, bloodmay be withdrawn from the subject followed by subjecting the withdrawnblood to said depletion of immunoglobulins after which the blood issubsequently administered back to said subject. Immunoadsorption of abody fluid can be carried out separate from the body of a subject, e.g.a human. Immunoadsorption of a body fluid can also be carried outcontinuously. For example, the body fluid such as blood may becontinuously withdrawn from a vein, the blood (e.g. whole blood or acomponent thereof) subsequently subjected to immunoadsorption, andre-infused back to the subject (e.g. (reconstituted blood). As shown inthe examples, such methods also may include the separation of the bloodinto blood plasma and a fraction comprising cellular components, whereinthe blood plasma is subjected to immunoadsorption, after which theobtained blood plasma that has been subjected to immunoadsorption isrecombined with the cellular components and infused back to the subject(see FIG. 1A). With regard to depletion of immunoglobulins, it isunderstood to comprise a reduction of the concentration of theimmunoglobulins.

For example, the total amount of immunoglobulins may be reduced at leasttwofold. Preferably, the total amount of immunoglobulins is reduced atleast fourfold, eightfold, tenfold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold,250-fold, 300-fold, 500-fold, 1000-fold, or more. The fold reduction ofimmunoglobulins may be detected by measuring one of the subclasses ofimmunoglobulins, e.g. IgA, IgD, IgE, IgG or IgM in the body fluid, e.g.blood serum or plasma, representative of the total amount ofimmunoglobulins. In general in serum, about 80% of immunoglobulins isIgG, 15% is IgA, 5% is IgM, 0.2% is IgD and a trace is IgE. The foldreduction of immunoglobulins may also be detected by measuring only IgGin the body fluid, e.g. blood serum or plasma, which can be regarded tobe representative of the total amount of immunoglobulins. The foldreduction of immunoglobulins may also be detected by measuring at leastone of IgG, IgA and IgM in the body fluid, e.g. blood serum or plasma,which is representative of the total amount of immunoglobulins. The foldreduction of immunoglobulins may also be detected by measuring all ofthe subclasses of immunoglobulins, e.g. IgA, IgD, IgE, IgG or IgM in thebody fluid, e.g. blood serum or plasma, representative of the totalamount of immunoglobulins. For example, from blood plasma or serum, theamount of IgG is measured before immunoadsorption and compared with theamount of IgG after immunoadsorption to calculate the fold reduction ofIgG, which can be representative for the fold reduction achieved usingimmunoadsorption. Serum immunoglobulins (IgG, IgA and IgM) levels cane.g. be determined by a commercial nephelometry assay using a BN-IIdevice (Dade Behring, Marburg, Germany). The manufacturer indicates thefollowing reference intervals for healthy adults: IgA 70-400 mg/dl, IgG700-1600 mg/dl and IgM 40-230 mg/dl (Dati et al., Eur J Clin Chem ClinBiochem. 1996; 34:517-20.

Alternatively, the AAV neutralizing antibody titer and/or AAV bindingantibody titer may be determined before and after immunoadsorption. Asshown in the example section, 3 cycles of immunoadsorption can result inan up to 16-fold reduction of AAV neutralizing antibody titer (see FIGS.2A-2B). Without being bound by theory, 4 cycles of immunoadsorption canreduce the AAV neutralizing antibody titer about 40-fold, whereas 5cycles of immunoadsorption can reduce AAV neutralizing antibody titer upto 100-fold. AAV neutralizing antibody titer and AAV binding antibodyassays are well known in the art and the skilled person is well capableof performing such assays (such as i.a. described by Ito et al., AnnClin Biochem 2009; 46: 508-510). Briefly, AAV neutralizing antibodytiters are determined by measuring the residual expression of a(reporter) gene in cells after transduction with an rAAV vector that hasbeen pre-incubated with a dilution series of a test-serum. The output ofsuch an assay can be expressed as the first dilution at which 50% orgreater inhibition of the reporter signal is detected (e.g. amount ofcells transduced and/or amount of expressed transgene). AAV bindingantibody titer can be determined with an ELISA. Briefly, microtiterplates can be coated with intact particles of recombinant AAV vectorswhich are incubated with dilution series of test-serum, followed byincubation with a horseradish peroxidase-conjugated anti-humanimmunoglobulin G (HRP-IgG). It is understood that it is not required todetermine the fold reduction of immunoglobulins, neutralizing antibodytiter or AAV binding antibody titer in a subject that is to have rAAVadministered. Nevertheless, one may monitor the fold reduction in asubject and once the desired reduction in e.g. immunoglobulins isachieved the rAAV vector can be administered.

It is understood that the number of cycles of immunoadsorptioncorrelates to fold reduction in immunoglobulins, either measured bydetermining the fold reduction of a particular immunoglobulin subclassor specific immunoglobulin titer such as AAV binding/neutralizingantibodies. Hence, the skilled person is well capable of determining theconditions such as the number of cycles required to achieve a certainreduction of immunoglobulins in any subject. With regard to the numberof cycles of immunoadsorption, it is understood that with regard to theprocessing of blood plasma 1 cycle corresponds to the processing of a 1volume of blood plasma as calculated according to Sprenger et al.(Sprenger, et al. J Clin Apher. 1987; 3: 185-190), which is incorporatedby reference herein in its entirety. Hence, in another aspect of theinvention, immunoadsorption comprises at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9 or atleast 10 cycles of immunoadsorption.

With regard to depletion of immunoglobulins using immunoadsorption it isunderstood that such a method is not a method of plasma exchange. Assaid, plasma exchange methods are widely known in the art and have beensuggested and used in rAAV vector administrations. Plasma exchangeinvolves the continuous withdrawal of blood from a vein, which blood isanticoagulated, after which it is separated into its liquid, i.e.plasma, and cellular components by e.g. hemofiltration. The separatedplasma in plasma exchange is replaced either by plasma from a donor(e.g. not having neutralizing antibodies) or by a defined replacementfluid such as e.g. a saline solution with 5% albumin. Thatimmunoadsorption is not plasma exchange does not exclude to usedepletion of immunoglobulins by using immunoadsorption in combinationwith plasma exchange. Hence, as long as the subject has been subjectedto depletion of immunoglobulins by immunoadsorption, the use of an rAAVvector in accordance with the invention is contemplated.

Furthermore, the application of immunoadsorption according to theinvention does not exclude to use depletion of immunoglobulins by usingimmunoadsorption in combination with other interventions. For example,subjects may also be subjected to immune suppression regimens well knownin the art that are aimed at reducing immune responses, which caninclude reducing the humoral response, induced by treatment with rAAV.Subjects may also be subjected to strategies to induce immune toleranceto AAV capsids. Hence, the subjects that are to have rAAV administeredand are to be subjected to the extracorporeal treatment may be subjectedto other concomitant interventions that are also aimed at reducing thehumoral immunity and/or cellular immunity. Such interventions may forexample be used in a first treatment with rAAV, wherein in anysubsequent treatment immunoadsorption in accordance with the inventionmay be applied. In subsequent treatments such interventions may beapplied as well. As said, immunoadsorption in accordance with theinvention is defined as the use of a binding moiety that is capable ofbinding immunoglobulins such that the immunoglobulins bound to thebinding moiety can be separated from the blood, preferably from theblood plasma. Such a binding moiety may be any kind of binding moiety,as long as it is capable of selectively binding to immunoglobulins. Suchbinding moieties preferably bind at least one of IgA, IgD, IgE, IgG andIgM. Such binding moieties may bind IgA, IgD, IgE, IgG or IgM. In oneembodiment the binding moieties may bind predominant immunoglobulinsthat are present in the blood, such as IgG, IgA and IgM, most preferablyIgG. It is understood that a binding moiety or binding moieties for oneor more immunoglobulins may be selective, e.g. IgG may be bound by abinding moiety whereas the other immunoglobulins are not substantiallybound or IgG, IgA and IgM are bound whereas the other immunoglobulinsare not substantially bound. Preferably however such binding moietiesbind the immunoglobulins IgA, IgD, IgE, IgG and IgM. It is alsounderstood that the binding moieties in accordance with the inventionmay be selected that also bind immunocomplexes, i.e. immunoglobulinsbound to antigens. Such binding moieties may include one or more bindingmoieties selected from the group consisting of peptides (such as e.g.Gam-146 and phenyl-alanine), dextran sulfate, tryptophan, protein A,protein G, protein A/G, protein L, and anti-human immunoglobulinantibodies. In one embodiment the binding moiety is a protein or apeptide or an aptamer.

The binding moieties according to the invention are preferably attachedto a matrix. Such attachment or coupling to a matrix may be covalent ornon-covalent. Suitable matrices are e.g. cellulose, sepharose, silica,cellulose, and polyvinyl-ethanol. Many suitable matrices are known inthe art as well as suitable methods to covalently or non-covalentlyattaching the binding moieties thereto. The binding moieties attached toa matrix allows contacting e.g. of blood plasma therewith to allowbinding of immunoglobulins thereto and separate immunoglobulins bound tosaid binding moiety from the blood plasma. Hence, a suitable bindingmoiety non-covalently or covalently attached to the matrix in accordancewith the invention is selected such that the binding moiety remainsattached to the matrix during interaction with blood plasma and/or serumor the like.

The binding of the immunoglobulin may be carried out by using a columnor a device. The term device or column can be used interchangeably andis understood to mean any suitable carrier to comprise a binding moietyor a matrix with binding moiety in which the binding of theimmunoglobulin, i.e. immunoadsorption, is to take place. Hence, saidcolumn or device preferably comprises a matrix with binding moiety. Theamount of matrix with binding moiety that is used provides for asufficient binding capacity to bind immunoglobulin. It is understoodthat sufficient binding capacity is easily attained by e.g. using atwo-column setup as shown in the examples with the commerciallyavailable system LIFE 18 Apheresis Unit (item nr. 330-000-098, MiltenyiBiotec GmbH, Bergisch Gladbach, Germany), wherein one column is beingregenerated while the other is processing plasma (see FIG. 1A). Such acolumn can comprise filters at either end to prevent particulate matterfrom entering or leaving the column. Hence, when the blood of a subjectis separated into plasma and cellular components, the plasma fractionflows through the column. The plasma and/or blood also be processed intoserum by removing clotting proteins. Anti-clotting agents may also beadded. Removal of clotting proteins from plasma and/or addinganti-clotting agents (e.g. heparin) may improve flow through the columnand/or interaction with the binding moieties. The plasma or serum isthus passed through such a column in accordance with the invention andthe plasma or serum collected while immunoglobulin bound to the bindingmoiety is retained in the column. After the plasma or serum from whichimmunoglobulins are depleted is collected, the blood can bereconstituted and returned to the subject.

Commercially available devices, or columns, that are suitable for theinvention include Immunosorba® (Fa. Fresenius HemoCare, St. Wendel)which uses as a binding moiety staphylococcal protein A and hasSepharose as a matrix; Prosorba® (Fa. Fresenius HemoCare, St. Wendel)which has as a binding moiety staphylococcal protein A and silica as amatrix; Globaffin® (Fa. Fresenius HemoCare, St. Wendel) which uses animmobilized synthetic peptide GAM® as a binding moiety with a SepharoseCL-4B matrix, which binding properties similar to those of the proteinA; Therasorb® Ig flex (Miltenyi Biotex, Bergisch Gladbach, Germany)which uses polyclonal sheep anti-human Ig with Sepharose CL-4B as amatrix; Selesorb® (Kaneka Medical Products, Osaka Japan), which uses adextran sulfate binding moiety and a cellulose gel as a matrix;Immusorba® (Fa. ASAHI/Diamed, Cologne) are based on tryptophan (TR-350L)or phenylalanine binding moiety (PH-350L), which are attached to apolyvinylethanol-gel matrix. Such commercially available columns, or thelike, can be well used in accordance with the invention, for example ina two-column device like the LIFE 18 Apheresis Unit (Miltenyi) or theArt Universal (Fresenius Medical Care).

In one embodiment, the binding moiety in accordance with the inventionis a binding moiety that is capable of binding anti-AAV immunoglobulins.It is understood that such a binding moiety may be specific for anti-AAVimmunoglobulins. Hence, in a further embodiment such a binding moiety iscapable of binding specifically anti-rAAV immunoglobulins. With regardto specificity of binding moieties it is understood that the majority ofimmunoglobulins will not be specific for AAV, hence, by selectivelybinding anti-AAV immunoglobulins the circulating antibody repertoire isto remain largely intact with the exception of anti-AAV immunoglobulinswhich are to be depleted. Selectivity of a binding moiety can easily bedetermined with immunoglobulin assays such as described above. Forexample, the total amount of immunoglobulins (or a subgroup thereof) maynot be significantly affected by immunoadsorption whereas the AAVbinding/neutralizing antibodies are reduced. It is understood that inthis embodiment such a binding moiety preferably is a protein, as AAVcapsids comprise protein. For example, AAV epitopes which are known tobe capable of generating anti-AAV antibodies can be used as bindingmoieties. Such epitopes can be attached to a suitable matrix and bepresented such to mimic binding sites as present in an AAV capsid. Also,AAV capsid proteins, such as VP1, VP2 and/or VP3, may be used as bindingmoieties. AAV capsids may also be used as binding moieties for bindinganti-rAAV immunoglobulins. Preferably, the peptides, AAV proteins or AAVcapsids used as binding moieties for binding immunoglobulins are derivedfrom the AAV serotypes which is to be administered. It is understoodthat AAV capsids comprise empty capsids and/or full capsids. It is alsounderstood that AAV capsids used as binding moieties may also be theactual product that is to be administered to the subject. The bindingmoieties capable of binding anti-rAAV immunoglobulins, as for any otherbinding moiety as described above, may be attached to any appropriatematrix. As said, such attachment may be covalent or non-covalent. Asuitable non-covalent attachment may include the binding of a suitableAAV capsid via an AAV specific antibody (such as a nanobody). Forexample, a chromatography resin commercially available for binding AAVof at least serotypes 1, 2, 3 and 5 is AVB Sepharose High Performance(GE Healthcare Bio-Sciences, Pittsburgh, USA, i.a. product code28411202). Hence, AAV capsids bound to this resin may be used as asuitable binding moiety/matrix in accordance with the invention. Also,e.g. for AAV5 capsids, a sialic-acid-rich protein called mucincovalently attached to a sepharose was shown suitable to bind AAVcapsids non-covalently (Mol Ther. 2001 October; 4(4):372-4). Althoughsuch capsids are not covalently bound to said matrices, such capsids maynot easily be released from the matrix. This is because high saltconcentrations and/or extreme pH values are required to release thecapsids, which extreme concentrations and pH values are not compatiblewith blood plasma and/or serum. Hence, a binding moiety non-covalentlyattached to a matrix is selected such that the binding moiety remainsattached during interaction with blood plasma and/or serum or the like.In addition, as the subject is to be administered with a correspondingAAV product any minor leakage from such a non-covalently attached AAVcapsid in addition to the later on administered AAV product does notprovide any risk from a safety point of view as said capsid is alsocomprised in the administered AAV product. Capsids may also becovalently attached to cyanogen-bromide-activated Sepharose 4B (GEHealthcare Bio-Sciences, Pittsburgh, USA, i.a. product code 17-0430-01)via —NH₂ groups.

In any case, such AAV specific binding moieties attached to a matrix maybe used in accordance with the invention as described above. Suchimmunoadsorption advantageously resulting in reducing AAVbinding/neutralizing antibodies while retaining most of the otherimmunoglobulins. In addition, these may also be used in a combinedfashion. For example, the body fluid may first be subjected toimmunoadsorption that is selective for immunoglobulins, followed by asecond immunoadsorption that is selective for immunoglobulins that canbind AAV capsids which may be in series or in parallel (see FIGS. 1B and1C). The body fluid may also be first subjected to immunoadsorption thatis selective for immunoglobulins that can bind AAV capsids followed by asecond immunoadsorption that is selective for immunoglobulins, or onecan even envisage both types of immunoadsorption to be carriedsimultaneously. In any case, it is preferred to have the body fluidfirst subjected to immunoadsorption that is selective forimmunoglobulins, followed by a second immunoadsorption that is selectivefor immunoglobulins that can bind AAV capsids. The firstimmunoadsorption reduces the total amount of immunoglobulins in the bodyfluid, e.g. blood serum or plasma, whereas the second immunoadsorptionreduces AAV specific immunoglobulins that remain. Hence, advantageously,the total AAV binding/neutralizing antibody titer can be reduced evenfurther. Hence, in another embodiment, an rAAV vector is provided foruse in the treatment of a subject according to the invention, whereinsaid immunoadsorption comprises two immunoadsorptions, oneimmunoadsorption with a binding moiety that binds immunoglobulins andone immunoadsorption that comprises a binding moiety that bindsanti-rAAV immunoglobulins. Said two immunoadsorptions may comprise oneimmunoadsorption with a binding moiety that binds selectivelyimmunoglobulins and one immunoadsorption that comprises a binding moietythat selectively binds anti-rAAV immunoglobulins.

With regard to the treatment of a subject, said subject may be a subjecthaving (or being suspected to have) AAV binding/neutralizing antibodiesprior to said extracorporeal depletion of immunoglobulins usingimmunoadsorption. This may be because the subject has been treated withan rAAV vector before, or because the subject has been exposed towild-type AAV. The presence of AAV binding and/or neutralizingantibodies may be determined using an assay such as described in theexample section. AAV binding antibody titers can be measured usingELISA. Briefly, AAV capsid proteins are immobilized on polystyrene ELISAplates and incubated with dilution series of serum samples to be tested.Bound antibodies can subsequently be detected by incubation withconjugated antibodies against immunoglobulins bound to AAV capsidproteins. rAAV neutralizing antibody titer can be determined by using acell-based assay, in which rAAV is pre-incubated with a dilution seriesof a serum sample after which cells are infected with said pre-incubatedrAAV and transduction efficiency and/or transgene expression isdetermined. The output of such an assay typically is the dilution atwhich less than 50% of transduction efficiency and/or transgeneexpression is detected, as compared with a control (e.g. un-incubatedrAAV or rAAV pre-incubated with control serum). As can be observed inthe example section, the detection of neutralizing antibodies can varywith regard to its sensitivity which may differ when e.g. differentcells are used between assays (see FIGS. 2A and 2B and take note at thescale of the y-axis). Nevertheless, the trends observed between assayscan be similar. It is understood that it may not be necessary to carryout any assays on body fluids obtained from a subject for detection ofsuch binding/neutralizing antibodies, as it can also be envisaged thatin any case, the depletion or reduction of rAAV binding/neutralizingantibodies in accordance with the invention generally obtained can besufficient to allow administration of rAAV to any subject in apopulation. Nevertheless, it may be preferred to carry out an assay fordetermining the rAAV binding/neutralizing antibody titer prior to rAAVadministration. Hence, in another embodiment, the rAAV vector for a usein the treatment of a subject according invention, comprises thetreatment of a subject having neutralizing antibodies for the capsid ofsaid rAAV vector prior to said extracorporeal treatment. Also, inanother embodiment, the rAAV vector for a use in the treatment of asubject according invention, comprises the treatment of a subject havingbinding antibodies for the capsid of said rAAV vector prior to saidextracorporeal treatment.

In one embodiment, the rAAV vector for use in the treatment of a subjectaccording invention, wherein said treatment comprises a subject havingneutralizing antibodies for the capsid of said rAAV vector prior to saidextracorporeal depletion of immunoglobulins as described above. Inanother embodiment, the rAAV vector for use in the treatment of asubject according to invention, comprises the treatment of a subjecthaving received a previous treatment with an rAAV vector. Hence, in oneembodiment an rAAV vector is provided for use in the treatment of asubject, wherein said subject has been subjected to an extracorporealdepletion of immunoglobulins using immunoadsorption prior toadministration of said rAAV vector, and wherein said subject hasreceived a previous treatment with an rAAV vector. Preferably, the rAAVserotypes used in the previous treatment and subsequent treatment arethe same. Preferably, the rAAV capsids used in the previous treatmentand subsequent treatment are the same. In another embodiment, the capsidproteins of the rAAV vector of the first administration and the capsidproteins of the rAAV vector of the second administration are differentfrom each other. Preferably, the previous and subsequent treatment, i.e.the first and second treatment, are at least 1 year apart. It is alsounderstood that the subsequent rAAV vector administrations may alsocomprise more than two administrations. The subsequent rAAV vectoradministrations may comprise at least two subsequent administrations, atleast three subsequent administrations and so forth.

Advantageously, it was found that by applying immunoadsorption todeplete immunoglobulins from a subject, the delivery of rAAV to thetarget cell, i.e. transduction of the target cell, a similar or the samedose could be used in a subject as compared with the dose used in asubject being naïve (i.e. unexposed) to the corresponding AAV capsidserotype. In another embodiment, the rAAV vector for use in thetreatment of a subject in accordance with the invention comprises thesame dose of rAAV vector as compared with the dose administered to asubject not having neutralizing antibodies for said capsid. In a furtherembodiment, the rAAV vector for use in the treatment of a subject inaccordance with the invention comprises a dose of rAAV vector which isat most 2-fold higher as compared with the dose for a subject not havingneutralizing antibodies for said capsid.

Without being bound by theory, it is understood that the extracorporealdepletion of immunoglobulins may also be advantageous in subjects thatdo not necessarily have any detectable neutralizing antibodies. As canbe observed in the example section, the detection of neutralizingantibodies involves a biological assay which can vary with regard to itssensitivity. Nevertheless, regardless of any neutralizing antibodiesdetected, antibodies present in the blood, or another bodily fluid, maystill interact with rAAV thereby hampering delivery to the target cells.Hence, in another embodiment, the extracorporeal depletion ofimmunoglobulins may advantageous improve the potency of the rAAV vectorbeing administered. Potency being defined as the number of cells beingtransduced and/or the amount of transgene expressed in the cell. Hence,without being bound by theory, the number of cells being transducedand/or the amount of transgene being expressed in the cell may beimproved in a subject that has been subjected to extracorporealdepletion of immunoglobulins prior to administering the rAAV vector. Inanother embodiment, the rAAV vector for use in the treatment of asubject in accordance with the invention comprises a dose of rAAV vectorwhich is lower as compared with the dose for a subject not having anydetectable neutralizing antibodies for said capsid. In a furtherembodiment, said dose is at least 20%, at least 30%, at least 40% or atleast 50% or more lower as compared with the dose required to achieve adefined transduction level in a subject not being subject to theextracorporeal depletion of immunoglobulins. A desired transduction canbe defined e.g. as resulting a certain expression level in the subjectby measuring the amount of transgenic protein detected in e.g. theblood, or any other appropriate measurement known in the art todetermine transduction in a subject.

In one embodiment, the rAAV vector for use in the treatment of a subjectaccording to the invention wherein the administration is selected fromthe group consisting of an intravenous administration, an intramuscularadministration, intra-hepato arterial administration, portal veinadministration, intraperitoneal administration, intracoronaladministration. Routes of administration that are in particular usefulare routes wherein neutralizing antibodies may be encountered uponadministration. Such routes may include an administration route whereinthe rAAV vector is exposed to blood and/or cerebrospinal fluid. It isunderstood that such a route of administration may also compriseadministration to the central nervous system. Neutralizing antibodiesmay for example hamper intrathecal administration of rAAV orstereotactic injections in the brain and/or spinal cord when e.g. theblood brain barrier function is compromised.

In another embodiment, the rAAV vector for a use in the treatment of asubject in accordance with the invention, comprises an extracorporealimmunoadsorption that is at most 24 hours prior to administration ofsaid rAAV vector. Preferably the time period between said rAAV vectoradministration and said extracorporeal immunoadsorption (also referredto as extracorporeal depletion of immunoglobulins usingimmunoadsorption) is short. Preferably, the said rAAV vectoradministration and extracorporeal immunoadsorption are at most 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 hours apart. Preferably said rAAV vector administration andextracorporeal immunoadsorption are performed sequentially at most 50,40 or 30 minutes apart. Most preferably said rAAV vector administrationand extracorporeal immunoadsorption are performed sequentially withoutany substantial pause, i.e. immediately following the extracorporealimmunoadsorption procedure said rAAV vector is to be administered.

Accordingly, in accordance with the current invention as describedabove, a method is provided for reducing the anti-rAAV immunoglobulinconcentration in a body fluid, comprising the steps of:

a) providing a body fluid;

b) providing a device for immunoadsorption;

c) subjecting the body fluid to immunoadsorption for anti-rAAV usingsaid device.

It is understood that a body fluid in accordance with the invention maybe any kind of body fluid, i.e. blood plasma, or blood serum, orcerebrospinal fluid. Hence, in accordance with the current invention asdescribed above, a method is also provided for reducing the anti-rAAVimmunoglobulin concentration in the blood, comprising the steps of:a) providing blood;b) providing a device for immunoadsorption;c) separating the blood provided in a) in plasma components and cellularcomponents;d) subjecting the plasma components obtained in c) to immunoadsorptionby using the device provided in b);e) reconstituting the blood by combining the cellular componentsobtained in c) with the plasma components subjected to immunoadsorptionobtained in d).In accordance with the invention also a method is provided for reducingthe anti-rAAV immunoglobulin concentration in the blood of a subject,comprising the steps:a) providing blood of the subject;b) providing a device for immunoadsorption;c) separating the blood provided in a) in plasma components and cellularcomponents;d) subjecting the plasma components obtained in step c) toimmunoadsorption by using the device provided in step b);e) reconstituting the blood by combining the cellular componentsobtained in c) with the plasma components subjected to immunoadsorptionobtained in d);f) administering the reconstituted blood obtained in step e) to thesubject. Such method may comprise in addition the step g) comprisingadministering to the subject an rAAV vector.

The transgene contained within the viral vector may not be a limitationof this invention. The invention is anticipated to be useful with anytransgene. Suitable transgenes for delivery to a patient in a viralvector for gene therapy may be selected by those of skill in the art.These therapeutic nucleic acid sequences typically encode products (e.g.proteins or RNA) for administration and expression in a patient in vivoor ex vivo to treat an inherited or non-inherited genetic defect, e.g.by replacing or correcting deficiency, to treat an epigenetic disorderor disease, or to treat a condition associated with dysregulation of agene product. Such therapeutic genes which are desirable for theperformance of gene therapy include, without limitation, a very lowdensity lipoprotein receptor gene (VLDL-R) for the treatment of familialhypercholesterolemia or familial combined hyperlipidemia, the cysticfibrosis transmembrane regulator gene (CFTR) for treatment of cysticfibrosis, DMD Becker allele for treatment of Duchenne musculardystrophy, and a number of other genes which may be readily selected byone of skill in the art to treat a particular disorder or disease. In apreferred embodiment, the rAAV vector comprises a transgene whichencodes a therapeutic protein, or an RNA, such as an miRNA. Preferably,the therapeutic protein is selected from the group consisting of factorIX (preferably human factor IX), factor VIII (preferably human factorVIII), lipoprotein lipase (LPL; including mutants such as for exampleLPL^(S447X); see WO 01/00220 A2), porphobilinogen deaminase (PBGD), verylow density lipoprotein receptor (VLDL-R), cystic fibrosis transmembraneconductance regulator (CFTR), Duchenne muscular dystrophy (DMD) Beckerallele, hypoxyluria (AGXT), N-acetyl-alpha-D-glucosaminidase (NaGlu),glial cell line-derived neurotrophic factor (GDNF), S100A1 (also knownas S100 calcium-binding protein A1, which in humans is encoded by theS100A1 gene). In a preferred embodiment, the therapeutic protein isfactor IX, more preferably human factor IX.

Alternatively, or in combination with any one of the precedingembodiments, in a preferred embodiment, the gene therapy is fortreating, preventing, curing and/or reverting a condition or disease,preferably a so-called orphan disease, which is herein understood to bea rare disease that affects a small percentage of the population, e.g.fewer than 1 in 1,500 people of the population that is life-threatening,chronically debilitating and/or inadequately treated. Generally, anorphan disease is a genetic disease and hence a life-long disease evenif symptoms do not immediately appear. In a preferred embodiment suchcondition or disease is selected from the group consisting oflipoprotein lipase deficiency (LPLD), hemophilia B, acute intermittentporphyria (AIP), Sanfilippo B syndrome, Parkinson's Disease (PD),congestive heart failure (CHF), Hemophilia A, Huntington's disease,Duchenne Muscular Dystrophy (DMD), Leber's congenital amaurosis,X-linked severe combined immunodeficiency (SCID), adenosine deaminasedeficiency severe combined immunodeficiency (ADA-SCID),adrenoleukodystrophy, chronic lymphocytic leukemia, acute lymphocyticleukemia, multiple myeloma, cystic fibrosis, sickle cell disease,hyperlipoproteinemia type I, thalassemia, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), epilepsy, Friedreich's ataxia,Fanconi anemia, Batten disease, wet AMD, alfa-antitrypsin-1, Pompedisease, SMA-1, Drug-resistant non-small cell lung cancer, GM1gangliosidosis, retina pigmentosa, homozygous FamilialHypercholesterolemia, lysosomal storage diseases, a copper or ironaccumulation disorders (e.g., Wilson's or Menkes disease), lysosomalacid lipase deficiency, hypoxyluria, Gaucher's disease, Hurler'sdisease, adenosine deaminase deficiency, glycogen storage disease and aretinal degenerative disease (such as RPE65 deficiency, choroideremia).

Alternatively, or in combination with any one of the precedingembodiments, in a preferred embodiment, the rAAV vector compositionfurther comprises a pharmaceutically acceptable carrier, diluents,solubilizer, filler, preservative and/or excipient. The rAAV vectorbearing a therapeutic gene may be administered to a patient, preferablysuspended in a biologically compatible solution or pharmaceuticallyacceptable delivery vehicle. A suitable vehicle includes sterile saline.Other aqueous and non-aqueous isotonic sterile injection solutions andaqueous and non-aqueous sterile suspensions known to be pharmaceuticallyacceptable carriers and well known to those of skill in the art may beemployed for this purpose. The viral vector is administered insufficient amounts to transfect the desired cells and provide sufficientlevels of transduction and expression of the selected transgene toprovide a therapeutic benefit without undue adverse or with medicallyacceptable physiological effects which can be determined by thoseskilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include direct delivery to thetarget organ, tissue or site, intranasal, intravenous, intramuscular,subcutaneous, intradermal, oral and other parental routes ofadministration. Routes of administration may be combined, if desired.Dosages of the rAAV vector will depend primarily on factors such as thecondition being treated, the selected gene, the age, weight and healthof the patient, and may thus vary among patients. For example, atherapeutically effective human dosage of the rAAV for primoadministration is generally in the range of from about 20 to about 50 mlof saline solution containing concentrations of from about 1×10⁷ to1×10¹⁰ pfu/ml viruses. A preferred adult human dosage is about 20 mlsaline solution at the above concentrations. The dosage will be adjustedto balance the therapeutic benefit against any side effects. The levelsof expression of the selected gene can be monitored to determine theselection, adjustment or frequency of dosage administration.

In a further aspect, the present invention relates to use of an rAAVvector for the manufacture of a medicament for the treatment of adisease, condition or disorder as specified above.

In a further aspect, the present invention relates to a kit of partscomprising an rAAV vector as defined herein, a device forimmunoadsorption as defined herein. In a preferred embodiment, the kitfurther comprises instructions for use of the kit.

Each embodiment as identified herein may be combined together unlessotherwise indicated.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: Immunoadsorption set up. From a (human) subject blood iscontinuously withdrawn. Optionally, an anticoagulant (heparin) may beadded to the withdrawn blood. The blood is subsequently processed by aseparating device (A) separating the blood into plasma (c) and cellularconstituents (b). The plasma subsequently flows through animmunoadsorption column (C). The immunoadsorption set up can comprisetwo adsorption columns (B and C) that may be used in an alternatingfashion such as in the examples. For example, while one is used forplasma processing, the other column may be cleaned. This way, thebinding capacity of the columns may be used optimally. Alternatively,the columns B and C may be different types of columns that are used inan alternating fashion. The processed plasma (d) is subsequentlyrecombined with the cellular constituents (b) and infused back (e) tothe (human) subject.

FIG. 1B: Immunoadsorption set up. From a (human) subject blood iscontinuously withdrawn. Optionally, an anticoagulant (heparin) may beadded to the withdrawn blood. The blood is subsequently processed by aseparating device (A) separating the blood into plasma (c) and cellularconstituents (b). The plasma subsequently flows through animmunoadsorption column (C). The immunoadsorption set up can comprisetwo adsorption columns (B and C) used in an alternating fashion whereinwhen one is used for plasma processing, the other column is beingcleaned. The processed plasma (d) is subsequently passed through asecond column (D) which is different from columns B and C, e.g. animmunoadsorption specific/selective for anti-AAV immunoglobulins. Theprocessed plasma (f) is than recombined with the cellular constituents(b) and infused back (e) to the (human) subject.

FIG. 1C: Immunoadsorption set up. From a (human) subject blood iscontinuously withdrawn. Optionally, an anticoagulant (heparin) may beadded to the withdrawn blood. The blood is subsequently processed by aseparating device (A) separating the blood into plasma (c) and cellularconstituents (b). The plasma subsequently flows through animmunoadsorption column (C). The immunoadsorption set up can compriseseveral adsorption columns (B, C and D) used in an alternating fashionwherein when one is used for plasma processing, the other columns may becleaned. The columns used may be different or the same. For example,columns B and C may be used in an alternating fashion as shown in theexamples. Plasma processing may be switched to column D after or beforeone or more cycles of processing by the B/C columns. The processedplasma (d) is subsequently recombined with the cellular constituents (b)and infused back (e) to the (human) subject.

FIGS. 2A and 2B. Neutralizing antibody titer reduction duringimmunoadsorption. The neutralizing antibody titer was determined inplasma obtained from non-human primates (0108, 7028, 7310) at differenttime points. Each timepoint indicating one cycle of immunoadsorption.FIGS. 2A and 2B shows results from different neutralizing antibodytitration assays using PLC/PRF/5 cells or HEK293 cells, respectively.

FIG. 3. SEAP expression in NHP. AAV5 SEAP was administered at day 0.AAV5 hFIX was administered at day 49.

FIG. 4. human FIX expression in NHP. AAV5 hFIX was administered at day49. No hFIX expression was detected in non-human primates that were notsubjected to immunoadsorption. Initial hFIX expression levels were atthe same level as observed in naïve non-human primates.

EXAMPLES

AAV Vectors

Two different AAV vectors of the serotype 5 were used (AAV5). A firstvector encoded the secreted embryonic alkaline phosphates (SEAP)(AAV5-SEAP) and the second vector encoded human coagulation factor IX(hFIX) (AAV5-hFIX). Non-human primates (Macaca fascicularis, NHP, 3animals per group) tested negative for the presence of anti-AAV serotype5 neutralizing antibodies were used. The first administration ofAAV5-SEAP (1×10¹³ gc/kg) was at day 0. The second administration ofAAV5-hFIX (1×10¹³ gc/kg) was at day 49. At most 24 hours before thesecond administration immunoadsorption was performed.

Immunoadsorption

The immunoadsorption process was as follows. Blood was previouslycollected from the NHP in lithium-heparin tubes and stored at 4° C. forprocessing. After centrifugation at 3500 rpm for 5 min plasma wasdiscarded and 1 mL of SAG-mannitol per 4 mL of whole blood was added.The blood concentrate was used to reconstitute blood to fill the LIFE 18equipment volume for the immunoadsorption process (i.e. tubing,separator devices, immunoadsorption devices). The extracorporeal volumewas about 80 ml and the blood volume of the animals was estimated to beabout 8% of the bodyweight, i.e. for an animal of about 3 kg this wasassessed to be about 300 ml. Animals had received heparin prior to theimmunoadsorption process. Animals were cannulated at a vein and anartery and connected to the LIFE 18 equipment. Cellular components wereseparated from blood plasma using a filtration device LIFE 18 DiskSeparator (item nr. 330-000-038, Miltenyi Biotec GmbH, BergischGladbach, Germany). The plasma was subsequently passed through aTherasorb Ig flex column/device from Miltenyi (item nr. 330-000-462Miltenyi Biotex, Bergisch Gladbach, Germany) which uses polyclonal sheepanti-human Ig with Sepharose CL-4B as a matrix. The plasma that wassubjected to immunoadsorption was recombined with the cellularcomponents. Heparin, CaCl2 and plasmalyte was added and reconstitutedblood infused back to the animal.

Transgene Expression

The analysis of SEAP expression was performed with the SEAP ReporterGene Assay, chemiluminescent (Ref: 11 779 842 001) from Sigma. Thisassay allows the quantitation of SEAP in the serum samples. In eachplate, a negative (water) and a positive control (differentconcentrations serving as standard) was included. The SEAP concentrationwas calculated extrapolating from the SEAP standard curve. hFIXexpression was measured using ELISA plates (Nunc MaxiSorp plate. Ref:456537, Thermo Scientific) coated with 50 μL of anti-hFIX (AHIX-5041,HTI) diluted 1:3000 in carbonate buffer overnight at 4° C. The next dayplates were washed three times with PBS Tween20 (PBSt) and blocked withblocking solution (PBSt+6% BSA) for one hour at RT. Plates were washedthree times with 200 μL PBSt; All sera samples and all FIX standardswere diluted 1:100 in incubation buffer (2% BSA in PBSt). The dilutionswere added at a final volume of 100 μL and incubated for one hour at RT.All samples were tested in duplicate. All samples were tested induplicate. Plates were washed three times with PBSt, 100 μL ofHRPO-conjugated anti-hFIX were added (Ref: CL20040APHP, CedarlaneLaboratories, diluted 1:2000) and plates incubated for one hour at RT.They were then washed three times with PBSt and the reaction wasrevealed with TMB substrate. It was stopped after 30 min by addition ofH₂SO₄ 2N. The absorbance was read at 450 nm in a microplate reader. Thetotal antibody titre was calculated as the serum dilution with anabsorbance five-fold higher than the negative control.

Neutralizing Antibody (NAbs) Titer

The measurement of NAbs in serum was based on an in vitro assay usingAAV5 carrying the transgene luciferase (AAV5-luc) and the hepatic cellline PLC/PRF/5 (ATCC CRL-8024) or HEK293 cells. Transgene expression isrevealed by addition of luciferin. Cells were seeded into a 96-wellplate at a density of 10⁴ cells/well. NHP sera were prepared in DMEM/2%FBS in a total volume of 100 μL, beginning with a 1:4 dilution followedby a dilution series of 1:2. Cells were infected with 10⁶ AAV5 particlesand wild type (wt) adenovirus was added at an MOI of 1. AAV was dilutedin 100 μL DMEM/2% FBS and incubated with serial serum dilutions for 2 hat 37°. The mixture was then used to infect cells. Each serum dilutionwas tested in duplicate. Negative controls of non-infected cells as wellas positive controls of infected cells without NHP serum were includedin each plate. The infected cells were incubated for 48 h and luciferaseactivity was quantified. Light emission from each well was measured inphotons/cm2/seg.

Anti-AAV Antibody Titer

The quantification of total Abs against AAV5 was based on an ELISA assayusing the specific capsid to coat the plate. The presence of total Absspecific against each capsid is revealed using Protein A Peroxidase.ELISA plates (Nunc MaxiSorp plate. Ref: 456537, Thermo Scientific) werecoated with antigen (AAV5 cap) at 100 ng/well in carbonate bufferovernight to 4° C. The next day plates were washed three times with PBStween-20 (PBSt) to eliminate the rest of the antigen and blocked withblocking solution (PBS+3% FBS) to prevent unspecific binding. Afterwashing three times with 200 μL PBSt, serum dilutions in PBSt wereadded, starting with 1:9 followed by a dilution series of 1:3 in a finalvolume of 100 μL. All samples were tested in duplicate. Negativecontrols without serum were included in each plate. The serum dilutionswere incubated for 2 h at 37°. After this, the serum was removed, theplate was washed three times with PBSt, and 100 μL of protein Aperoxidase diluted 1:10,000 in blocking solution were added for onehour. The plate was washed three times with PBSt and the reaction wasrevealed with TMB substrate and stopped 30 min later with H₂SO₄ 2N. Theabsorbance was read at 450 nm in a microplate reader. The total antibodytitre was calculated as the serum dilution which had an absorbancefive-fold higher than the negative control. The total antibody titerreduction observed was about 9-fold, which was in line with thereduction observed in the Nab assay.

Ig, IgM, IgG1 Measurements

In order to facilitate the completion of an AAV re-administrationprocedure in the clinical setting, the total Immunoglobulin, IgM andIgG1 concentrations were measured before and after the immunoadsorptionprocedure. Measurements were carried out using assays developed forMacaca fascicularis. Reductions in general immunoglobulinsconcentrations observed were compatible with the Tab assay.

Results and Discussion

AAV neutralizing antibody titer and AAV binding antibody titer wasmeasured in the NHP throughout the entire experiment. The AAVneutralizing antibody titer and AAV binding antibody titer were in therange as observed in naïve subjects having AAV administered. Theimmunoadsorption process strongly reduced the neutralizing antibodytiter (FIGS. 2A-2B) in plasma. Importantly, after the second vectoradministration of AAV hFIX the animals that had receivedimmunoadsorption showed high levels of hFIX expression around 7 dayspost administration. hFIX declined 20 days post administration andremained stable thereafter (FIG. 4). Such an expression profile in timehas been described in previous NHP studies, also without prior exposureto AAV vectors and is independent of the serotype used. In contrast, theanimals that did not receive immunoadsorption completely lacked anydetectable hFIX expression at any time point. After the secondadministration a secondary response against AAV5 was observed by anincrease in neutralizing antibody titre and AAV binding antibody titre.Combined, the results show that immunoadsorption resulted in a stronglyreduced AAV neutralizing antibody titer in serum which provided for atime window that allows for highly efficient rAAV transduction.

Furthermore, measurements of Ig, IgM and IgG1 in the NHP related to NAband TAb assays. This confirms that commercially available assays thatare available for human Ig, IgM and/or IgG1 measurements may be used ina clinical setting to control the immunoadsorption and re-administrationprocedure. Such assays allow to determine the number of cycles required(by measuring immunoglobulin level before the immunoadsorptionprocedure), and/or allow to determine whether the desired reduction inimmunoglobulin concentration has been achieved (by measuring theconcentration shortly after the immunoadsorption procedure). Hence, theimmunoadsorption procedure can be adapted (e.g. determining the numberof cycles required) depending on immunoglobulin measurements in humansubjects to ensure successful re-administration. This may allow for awell-controlled procedure wherein both the immunoadsorption andre-administration are performed on the same day.

The invention claimed is:
 1. A method of administering a recombinantadeno-associated virus (rAAV) to a subject, comprising: (a) depleting asubject's circulating immunoglobulins by contacting the subject's bloodwith an extracorporeal device for immunoadsorption, the devicecomprising a binding moiety attached to a matrix, wherein the bindingmoiety binds immunoglobulins, and (b) subsequently administering a rAAVto the subject, wherein prior to depleting the subject's circulatingimmunoglobulins, the subject had neutralizing antibodies that bind to acapsid of the rAAV as a result of previously receiving a treatmentcomprising a rAAV vector.
 2. The method of claim 1, wherein the bindingmoiety is selective for one or more of IgA, IgD, IgE, IgG and/or IgM. 3.The method of claim 1, wherein the binding moiety is selected from thegroup consisting of peptides, dextran sulfate, tryptophan, protein A,protein G, protein A/G, protein L, and anti-human immunoglobulinantibodies.
 4. The method of claim 1, wherein the binding moietycomprises a protein that binds anti-rAAV immunoglobulins.
 5. The methodof claim 4, wherein the protein that binds anti-rAAV immunoglobulinsbinds to AAV VP1, AAV VP2, or AAV VP3.
 6. The method of claim 1, whereinthe rAAV is administered intravenously.
 7. A method of decreasing ahumoral immune response in response to a recombinant adeno-associatedvirus (rAAV) gene therapy in a subject comprising, (a) a firstimmunoadsorption step comprising contacting the subject's blood with afirst extracorporeal device comprising a binding moiety that bindsimmunoglobulins, wherein the binding moiety is attached to a matrix, (b)a second immunoadsorption step comprising contacting the subject's bloodwith a second extracorporeal device comprising a binding moiety thatbinds anti-rAAV immunoglobulins, wherein the binding moiety is attachedto a matrix, and (c) subsequently administering rAAV gene therapy to thesubject, thereby decreasing the humoral immune response to the rAAV genetherapy; wherein prior to depleting the subject's circulatingimmunoglobulins, the subject had neutralizing antibodies that bind to acapsid of the rAAV as a result of previously receiving a treatmentcomprising a rAAV vector.
 8. The method of claim 7, wherein the rAAV isadministered intravenously.
 9. The method of claim 7, wherein the firstand second immunoadsorption steps depletes the amount of neutralizingantibodies in the subject's blood by at least 90%.
 10. The method ofclaim 7, wherein the first and second immunoadsorption steps areperformed at most 24 hours prior to administration of the rAAV.
 11. Themethod of claim 7, wherein the subject is administered a dose of rAAVthat is the same as a dose for a subject not having neutralizingantibodies for a capsid of the rAAV.
 12. The method of claim 7, whereina cellular response the rAAV is reduced.
 13. A method for reducing ananti-rAAV immunoglobulin concentration in the blood of a subject,comprising: a) obtaining blood from the subject, wherein the subject'sblood comprises neutralizing antibodies that bind to a capsid of therAAV as a result of previously receiving a treatment comprising a rAAVvector; b) separating the blood into plasma components and cellularcomponents; c) contacting the plasma components with an extracorporealimmunoadsorption device, the device comprising a binding moiety attachedto a matrix, wherein the binding moiety binds anti-rAAV immunoglobulins;d) reconstituting the blood by combining the cellular components withthe plasma components that were subjected to immunoadsorption; and e)administering the reconstituted blood to the subject, thereby reducingthe anti-rAAV immunoglobulin concentration in the subject's blood. 14.The method of claim 13, further comprising administering an rAAV vectorto the subject.
 15. The method of claim 14, wherein the rAAV vector isadministered to the subject within 24 hours of administering thereconstituted blood to the subject.
 16. The method of claim 13, whereinthe anti-rAAV immunoglobulin concentration in the subject's blood isdepleted by at least 90%.